Single-molecule detection of transient dimerization of opioid receptors 1: Homodimers' effect on signaling and internalization

Abstract

Opioid receptors (ORs) are critical for endogenous and synthetic analgesics. Their homodimerization is considered important for their pharmacological diversities, but whether they form homodimers remains controversial. Here, we established that the three classical ORs, -, {kappa}-, and {delta}-ORs (MOR, KOR, and DOR, respectively) undergo repeated transient (120-180 ms) homodimerizations every few seconds. This was done by using single-molecule imaging and developing theories for analyzing single-molecule colocalization data, which provide the key parameters, homodimer-monomer dissociation equilibrium constants and rate constants. Their 9-26 amino-acid C-terminal cytoplasmic domains, without sequence similarities, are involved in specific homodimerization, whereas the transmembrane domains provide less specific affinities. Using the membrane-permeable peptides mimicking the C-terminal homodimerization sequences which block homodimerizations, functions of monomers and homodimers were dissected. KOR and DOR homodimers, but not MOR homodimers, activate downstream G-proteins differently from monomers upon agonist addition, without influencing OR internalization. These findings could guide strategies to enhance OR-based analgesia.